Our objective is to use adenovirus (Ad) as an "expression vector." We will introduce a foreign "passenger gene" (PG) into the Ad genome, and use the Ad-PG recombinant to prepare large quantities of the PG protein. We will concentrate on PG proteins that are important scientifically and pharmacologically, and that are difficult to obtain by conventional region E3 to form nondefective recombinants. PGs will be expressed late after infection under control of their own promoter and RNA processing signals; abundant PG protein synthesis will result from amplification of the PG gene by viral DNA replication. (ii) PGs will be fused on their 5' side to the hexon splice acceptor and on their 3' side to the hexon cleavage/polyadenylation signal, then inserted into E3 to produce nondefective recombinants. Expression of these PGs will be controlled by the very strong Ad major late promotor as well as by the cleavage/polyadenylation and splicing signals of the Ad hexon gene (high priority). The hexon gene was chosen because hexon is synthesized in much higher quantities than other Ad proteins and thus it probably has very strong RNA processing signals. Using a protocol that places PG expression under selective pressure, we will attempt to isolate regulatory "up" mutants tat synthesize enhanced levels of PG protein. (iii) PGs will be inserted into the hexon gene to produce defective recombinants. Our ultimate goal is to achievePG protein synthesis at the same level as hexon. We will use our Ad expression vector to prepare the transformation proteins coded by the Ela region of highly oncogenic Ad12 (first priority) as well as the Ela (second priority) and the Elb regions of Ad2. Chemical, physical, and biological characterization of these proteins will provide insights into Ad cell transformation.